The overall objective of this research project is to determine the role of apolipoprotein E (apoE) in Alzheimer's disease (AD). The present proposal is driven by the following hypotheses: (I) apolipoprotein E (apoE) interacts with amyloid precursor protein (APP) intracellularly; (II) both N-terminal and C-terminal domains of APP are involved in the interaction, and the binding site in the C-terminal domains of APP are involved in the interaction, and the binding site in the C-terminal domain is within the beta-amyloid protein (Abeta) sequence; (III) the direct interaction of apoE with APP affects the intracellular trafficking and re-internalization of APP and this, in turn, modifies the production of Abeta; and (IV) the association of apoE with Abeta facilitates the clearances of Abeta and thus, prevents Abeta aggregation. These hypothesis are based on a number of observations: 1) genetic evidence suggesting that apoE is a risk factor for AD, 2) apoE presence in association in the senile plaques and in complex with Abeta in cerebrospinal fluid and plasma, 3) the formation of a stable complex between apoE and full length APP as well as apoE and Abeta-containing C- terminal fragment of APP in co-transfected cells (preliminary data). The specific aims are: 1) to investigate whether the interaction between apoE and APP is isoform dependent and identify the apoE binding site(s) on APP. 2) to determine and compare the effects of each apoE isoform on APP processing, especially Abeta production. 3) to explore whether cell- derived Abeta is different from synthetic Abeta in binding to apoE, and evaluate the role of apoE in Abeta clearance. The research designs are as follows: a) the complex formation between apoE and APP will be analyzed in cells co-expressing apoE and APP; b) a series of deletion mutations on APP will be generated to map the apoE binding site(s); (c) the secretion and intracellular accumulation of Abeta and other derivatives of APP will be compared among the transfected cells; d) the subcellular compartments of proteolytic processing of APP will be identified by blocking transport at different subcellular sites or by tracking the radiolabeled cell surface APP; e) the interaction of apoE with (i) Abeta produced by transfected cells, (ii) Abeta obtained from AD brain, and (iii) synthetic Abeta, will be characterized; and f) the rate of degradation or clearance of metabolically labeled Abeta in conditioned medium will be assessed in the presence or absence of neural or non-neural cells transfected with apoE isoforms. The methods include: PCR, subcloning, metabolic labeling, biochemical labeling, immunoprecipitation and Western blot analyses. In conclusion, the proposed study will answer fundamental cellular and molecular biological questions related to the roles of apoE in Abeta amyloidogenesis and in AD by using a novel approach to investigate the role of apoE in APP metabolism and in Abeta clearance.